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Abstract:

This invention discloses GLP-1 analogues and their pharmaceutical salts,
wherein the GLP-1 analogue comprises an amino acid sequence of general
formula (I), wherein Lys represents a modified lysine with a lipophilic
acid. The GLP-1 analogues provided by this invention have the function of
human GLP-1, and a longer half-life in vivo compared with the human
GLP-1. Uses of such compounds and compositions include treating
non-insulin-dependent diabetes, insulin-dependent diabetes, and obesity.
TABLE-US-00001
(I)
X1-X2-Glu-Gly-Thr-Phe-Thr-Ser-Asp-X10-Ser-X12-
X13-X14-Glu-X16-X17-Ala-X19-X20-X21-Ph-
e-Ile-X24-
Trp-Leu-X27-X28-X29-X30-X31-X32-X33-X.-
sub.34-X35-X36-
X37-X38-X39-Lys

24. The GLP-1 analogue or a pharmaceutically acceptable salt thereof of
claim 23, wherein X1 is selected from L-His and D-His; X2 is
selected from Ala, D-Ala, Gly, Val, Leu, Ile, Lys and Aib; X10 is
selected from Val and Leu; X12 is selected from Ser, Lys and Arg;
X13 is selected from Tyr and Gln; X14 is selected from Leu,
Met, and Nle; X16 is selected from Gly, Glu and Aib; X17 is
selected from Gln, Glu, Lys and Arg; X19 is selected from Ala and
Val; X20 is selected from Lys, Glu and Arg; X21 is selected
from Glu and Leu; X24 is selected from Val, Lys, Glu, and Ala;
X28 is selected from Lys, Glu, Asn and Arg; X29 is selected
from Gly and Aibl; X30 is selected from Arg, Gly and Lys; X31
is selected from Gly, Ala, Glu, Pro and Lys; X32 is selected from
Lys and Ser; X33 is selected from Lys and Ser; X34 is selected
from Gly, Ala and Sar; X35 is selected from Gly, Ala and Sar;
X36 is selected from Pro and Gly; X37 is selected from Pro and
Gly; X38 is selected from Pro and Gly; X39 is selected from Ser
and Tyr.

25. The GLP-1 analogue or a pharmaceutically acceptable salt thereof of
claim 24, wherein the amide bond is formed by the lipophilic substituent
of formula R1(CH2)n--CO-- and an c amino group of the
C-terminal Lys residue.

32. The GLP-1 analogue or a pharmaceutically acceptable salt thereof of
claim 24, wherein the amide bond is formed by the lipophilic substituent
of formula R1(CH2)n--CO-- and an α amino group of
the C-terminal Lys residue.

Description:

FIELD OF THE INVENTION

[0001] This invention relates to analogues of the human Glucagon-like
peptide-1 (GLP-1) and pharmaceutical salts thereof. The GLP-1 analogues
provided in this invention have the function of the human GLP-1 peptide
and a longer half-life in vivo compared with the native protein. The
present invention also relates to the use of GLP-1 analogues, the
pharmaceutical salts thereof, or use as a pharmaceutical composition
thereof in the treatment of non-insulin-dependent diabetes,
insulin-dependent diabetes and obesity.

BACKGROUND OF THE INVENTION

[0002] Diabetes mellitus is a global epidemic disease and is a metabolic
disorder relating to glucose, protein and lipids due to the absolute or
relative deficiency of insulin (See Chen Ruijie. Status of research on
diabetes drugs. Academic journal of Guangdong College of Pharmacy, 2001,
7(2):131-133). Diabetes mellitus can be divided into type I diabetes
mellitus and type II diabetes mellitus (Type 2 diabetes mellitus, T2DM,
the same below) according to the pathogenesis thereof 90-95% of all the
patients diagnosed with diabetes mellitus suffer from T2DM, and patients
are often afflicted with obesity, a deficiency of physical activity. T2DM
is most common in the aging population, or among those with family
history of diabetes mellitus T2DM. It is also a progressive disease.
According to statistical data in 2000, the World Health Organization
estimated that there are about 171 million people worldwidely who suffer
from diabetes mellitus. In 2005, the U.S. Centers for Disease Control and
Prevention. estimated that 20.8 million Americans suffer from diabetes
mellitus which is about 7% of the population of the United States. In
2006, the International Diabetes Federation, estimated that the global
number of patients suffering from diabetes mellitus is about 246 million
(about 5.9% of the totally global population) and indicated that 46% of
the patients were 40-59 years old.

[0004] A major risk factor of T2DM is obesity, which is itself very
harmful to human health. T2DM often co-exists with other high-risk
diseases such as hypertension and dyslipidemia. 60% of T2DM patients are
accompanied by microvascular complications, including retinopathy and
neuropathy, and also are accompanied by cardiovascular morbidities, such
as coronary heart disease, myocardial infarction, shock, and the like. In
the U.S., cardiovascular diseases (CVD) is the major cause resulting in
mortality, and T2DM is the major risk factor causing macrovascular
complications such as an atherosclerosis, myocardial infarction, shock,
and peripheral vascular diseases. The risk of death caused by heart
diseases with diabetes is 2-4 times higher than that of a non-diabetes
person. In addition, nearly 65% of people with diabetes die of heart
disease.

[0005] In addition to the physical and physiological harm to patients,
T2DM causes great economic burden on society. According to statistics,
the cost of the treatment of complications associated with diabetes is
about $ 22.9 billion; the total cost of the treatment of T2DM and
complications thereof is nearly $ 57.1 billion every year in the U.S.

[0006] Drugs for the treatment of T2DM have been sought. These include the
early oral hypoglycemic drugs of sulfonyl class and biguanide class and
the recent insulin sensitizer and α-glucosidase inhibitors, the
development of animal insulins and human insulins in a variety of new
regimes and formulations, the research of new mechanisms of drug
treatment by simply increasing insulin, and new ways acting on the
insulin-producing cells. Weight gain is a common side-effect after the
administration of oral or injection hypoglycemic agents, which may reduce
compliance, and may increase the risk of developing cardiovascular
disease. Therefore, developing new types of drugs for the treatment of
T2DM which have high safety profiles, good patient compliance and low
side-effects is desirable.

[0007] As early as 100 years ago, Moore proposed that the duodenum can
secrete a "chemical stimulant" stimulating pancreatic secretion. Attempts
to inject gut-extract to treat diabetes were undertaken. Subsequently it
was discovered that humoral factors derived from intestinal secretion can
enhance the function of the pancreas endocrine, and about 50% of insulin
secretion induced by intravenous or oral glucose is derived from the
stimulus of peptides produced in the gut. Therefore Zunz and Labarre
described the concept of "incretin." Two kinds of incretins have been
isolated so far, namely glucose-dependent insulinotropic polypeptide
(GIP) and glucagon-like peptide-1 (GLP-1). Both GIP and GLP-1 are
secreted by specific intestinal nerve cells when a related nutrient is
absorbed. GIP is secreted by the duodenum and proximal jejunal K cells.
GLP-1 is synthesized in L cells and mainly exists in the distal small
bowel and colon (See Drucker D J. Enhancing incretin action for the
treatment of type 2 diabetes. Diabetes Care. 2003, 26(10):2929-2940).

[0008] GLP-1 exists in two bio-active forms in blood plasma, namely GLP-1
(7-37) and GLP-1 (7-36). The difference between the two forms resides in
one amino acid residue, and their biological effects and in vivo
half-life are the same. (See Drucker D J. Enhancing incretin action for
the treatment of type 2 diabetes. Diabetes Care. 2003, 26(10):2929-2940).

[0009] GLP-1 is usually referred to as GLP-1 (7-37) and GLP-1 (7-36)
amide. GIP and GLP-1 are degraded to inactive forms by dipeptidyl
peptidase-IV(DPP-IV) quickly after released in the gastrointestinal
tract, so that the in vivo half-life of GIP and GLP-1 is very short (in
vivo half-life of GIP is about 5-7 minutes, in vivo half-life of GLP-1 is
about 2 minutes). (See Drucker D J. Enhancing incretin action for the
treatment of type 2 diabetes. Diabetes Care. 2003, 26(10):2929-2940).
Researches show that most of the degradation process occurrs when the GIP
and GLP-1 enter into the blood vessels containing DPP-IV, and a small
amount of GLP-1 and GIP which has not been degraded will enter into the
pancreas and associate with binding sites to stimulate insulin release
from β-cells. Different from the mechanism of sulfonylurea to
directly promote functional β-cells to release insulin, most of the
effects of incretin are glucose-dependent. In addition, some in vitro
tests on animals and humans have shown that GLP-1 also functions to
suppress α-cell and reduce glucagon hypersecretion.

[0010] Although plasma GIP levels in patients with T2DM are normal, when
the function of incretin declines significantly, the GLP-1 levels in
patients with T2DM decline. Thus, drugs based on GLP-1 contribute more to
treatment of T2DM. Although the levels of both GLP-1 (7-37) and GLP-1
(7-36) amide will increase in several minutes after a meal, and the
content of GLP-1 (7-36) amide is more, so the GLP-1 secretion might have
been greatly increased by the double effect of endocrine and transmission
of neural signal before the digested food enters the small intestine and
colon. The plasma level of GLP-1 under a fasting state is very low (about
5-10 pmol/L), and is increased rapidly after eating (up to 15-50 pmol/L).
Under the double function of DPP-IV and renal clearance, the level in
vivo of GLP-1 in circulation is decreased rapidly. Other enzymes such as
human neutral endopeptidase 2411 may also play a vital role in
inactivating clearance of GLP-1. Because the second amino acid residue of
GLP-1 is alanine, which is a good substrate of DPP-IV, GLP-1 is easily
degraded into inactive peptide fragments. In fact, the DPP-IV in vivo is
postulated as the key reason for loss of the activity of the incretin.
Experiments show that GLP-1 levels in mice, in which DPP-IV gene has been
silenced, is higher than in normal mice. Significantly, insulin secretion
is increased, too. Just because the presence of DPP-IV, the content in
vivo (except in plasma) of the nondegradaded and biologically active
GLP-1 is only 10-20% of the total content of GLP-1 in plasma. (See Deacon
C F, Nauck M A, Toft-Nielsen M, et al. Both subcutaneously and
intravenously administered, glucagon-like peptide 1 is rapidly degraded
from the NH2-terminus in type 2-diabetic patients and in healthy
subjects. (See Diabetes. 1995, 44(9): 1126-1131).

[0011] GLP-1 and GIP play their respective roles through binding to
different G-protein-coupled receptors (GPCRs). Most of GIP receptors are
expressed by pancreatic β-cells, and a minor part of GIP receptors
are expressed by adipose tissue and the central nervous system. In
contrast, GLP-1 receptors are mainly expressed in the pancreatic α-
and β-cells and peripheral tissues including the central and
peripheral nervous systems, brain, kidney, lung and gastrointestinal
tract and the like. The activation of two incretins in β-cells will
result in the rapid increase of the level of cAMP and intracellular
calcium, thereby rleading to their extracellular secretion in a
glucose-dependent manner. The sustained signal transmission from incretin
receptors is associated with protein kinase A, resulting in gene
transcription, increasing insulin biosynthesis and stimulating
β-cell proliferation. (See Gallwitz B. Glucagon-like peptide-1-based
therapies for the treatment of type 2 diabetes mellitus. Treat
Endocrinol. 2005, 4(6):361-370). The activation of GLP-1 receptor and GIP
receptor can also inhibit the apoptosis of pancreatic β-cells of
rodent and human, while increasing their survival (See Li Y, Hansotia T,
Yusta B, et al. Glucagon-like peptide-1 receptor signaling modulates beta
cell apoptosis. J Biol. Chem. 2003, 278(1): 471-478). Consistent with the
expression of GLP-1 receptor, GLP-1 can also inhibit glucagon secretion,
gastric emptying and food intake, and enhance the degradation of glucose
through the neural mechanism. It shall be noted that, as with other
insulin secretion mechanisms, the role of GLP-1 to control the level of
glucose is glucagon-dependent and the counter-regulatory release of
glucagon caused by low blood sugar is fully retained even at the
pharmacological level of GLP-1.

[0012] The important physiological role of endogenous GLP-1 and GIP in
glucose homeostasis has been studied in-depth through using receptor
antagonists or gene knockout mice. Acute antagonism of GLP-1 or GIP
reduces insulin secretion in vivo of rodents and increases plasma glucose
content. Similarly, the mutant mice, in which GIP or GLP-1 receptor is
inactivated, also experience defective glucose-stimulated insulin
secretion and damaged glucose tolerance. GLP-1 also has a function of
regulating fasting blood glucose, because the acute antagonists or damage
on the GLP-1 gene will cause the increase of fasting glucose level of
rodents. At the same time, GLP-1 is the basis of glucose control in human
bodies, and studies on the antagonist of Exendin (9-39) have shown that
the destruction of GLP-1 function will result in defective
glucose-stimulated insulin secretion, decreased glucose clearance rate,
increased glucagon levels and accelerated gastric emptying. The
physiological roles of GLP-1 (see Deacon C F. Therapeutic strategies
based on glucagon-like peptide 1. Diabetes. 2004, 53(9):2181-2189)
comprise: (1) helping to organize glucose absorption, mediate
glucose-dependent insulin secretion; (2) inhibiting postprandial glucagon
secretion, reducing hepatic glucose release; (3) regulating gastric
emptying, preventing excessive circulating of glucose when the food is
absorbed in the intestine; and (4) inhibiting food intake (such as
appetite). Also, animal studies also showed a physiological role for
stabilizing the number of pancreatic β-cells in vivo.

[0013] Due to the beneficial effects of GLP-1 and GIP in controlling blood
sugar and many other aspects, especially their characteristics of not
producing hypoglycemia and delaying gastric emptying to control weight,
the compounds attract the interest of many scientists. Further studies of
based on GLP-1 and GIP for the treatment of T2DM have been pursued. It is
well known that T2DM patients lack or lose the incretin effect. One
reason is that incretin effect of GIP in vivo in the T2DM patient is
significantly reduced. Meanwhile, the level of GLP-1 in vivo in T2DM
patients is very low, and the level of GLP-1 caused by dietary stimuli is
significantly reduced. (See Toft-Nielsen M B, Damholt M B, Madsbad S, et
al. Determinants of the impaired secretion of glucagon-like peptide-1 in
type 2 diabetic patients. J Clin Endocrinol Metab. 2001,
86(8):3717-3723). Because the role of GLP-1 in vivo in patients with T2DM
has been partially reserved, GLP-1 synergist is one of the research
directions of the drugs designed to enhance the incretin effect in T2DM
patients.

[0014] GLP-1 analogues, may act similarly to endogenous GLP, by inhibiting
the release of glucagon and stimulate insulin secretion both in vivo in a
glucose-dependent manner and thus its role for lowering blood glucose
exhibit a self-limitation, which generally does not cause hypoglycemia in
large doses. Some literature reports that GLP-1 can reduce blood sugar to
a level below normal, and this effect is transient and considered a
natural result of GLP-1 promoted insulin secretion. GLP-1 can temporarily
reduce blood sugar to a level below normal level but does not cause
serious and persistent hypoglycemia. Besides directly reducing blood
glucose, GLP-1 can also reduce the quantity of food intake, which has
been verified in rodents and humans. The level of blood glucose,
therefore, can be controlled by reducing body weight indirectly. GLP-1
also has the potential role of inhibiting the secretion of gastrin and
gastric acid stimulated by eating, and these functions show that GLP-1
may also have a role in the prevention of peptic ulcer. Mechanisms of
action for GLP-1 make it an ideal drug for the treatment of patients with
type 2 diabetes, but also the drug for the treatment of patients with
obesity diabetes. GLP-1 can enhance the satiety of the patients, reduce
food intake and maintain body weight or lose weight. Several studies
suggest that GLP-1 can prevent the conversion from impaired glucose
tolerance to diabetes, and some literature reports that the GLP-1 class
of compounds has direct effect on the growth and proliferation of
pancreatic β-cells in experimental animals. It was found by some
experiments that GLP-1 can promote the differentiation from pancreatic
stem cells to functional β-cells. These results suggest that GLP-1
has the function of protecting pancreatic islet and delaying the
progression of diabetes, and can maintain the morphologies and functions
of β-cells, while reduce the apoptosis of β-cells. Because some
oral drugs and exogenous insulins can not inhibit or reduce the
exorbitant glucagon secretion in patients with T2DM, GLP-1 analogues can
affect glucagon hypersecretion through directly inhibiting glucagon
release or inhibition of glucagon resulted from promoting insulin
secretion. The postprandial hyperglycemia can be reduced effectively
through these two mechanisms. Meanwhile, the maintaining of the function
of β-cells may also play a role in controlling the long-term
postprandial hyperglycemia.

[0015] GLP-1 analogues are administered through subcutaneous injection,
which doesn't require calculation of the amount of carbohydrates to
estimate the optimal drug dosage, and does not require self-monitoring
the blood glucose. As a result, these kinds of drugs are easier for
patient compliance than self-administered insulin.

[0016] A variety of effects of natural GLP-1 have been confirmed, which
bring new hope for the treatment of T2DM. The natural human GLP-1 peptide
is, however, very unstable and can be degraded by dipeptidyl peptidase IV
(DPP-IV). Moreover, its half-life is only about 2 minutes. When using
natural GLP-1 to lower blood sugar, continuous intravenous infusion or
continuous subcutaneous injection is needed, resulting in its poor
clinical feasibility. Faced with this situation, researchers continue to
explore methods to extend the action time of GLP-1. Therefore, there is a
need for the development of long-acting GLP-1 analogues or derivatives
thereof.

[0017] Exenatide is a synthetic Exendin-4, which is developed by the Eli
Lilly Company and Amylin Company, with the trade name Byetta®.
Exenatide has been approved for the treatment of T2DM by FDA and EMEA. It
has 50% homology with mammalian GLP-1 in sequence and has a similar
affinity site of the receptor with GLP-1. (See Drucker D J, Nauck M A.
The incretin system: glucagon-like peptide-1 receptor agonists and
dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet. 2006,
368(9548):1696-1705). It is encoded by a lizard-specific gene. Compared
with GLP-1, the second residue, alanine, in GLP-1 is replaced with
glycine in Exenatide, which effectively inhibits the enzymolysis of
DPP-IV enzyme, and its half-life in vivo is about 60-90 minutes. (See
Kolterman O G, Kim D D, Shen L, et al. Pharmacokinetics,
pharmacodynamics, and safety of exenatide in patients with type 2
diabetes melllitus. Am Health Syst Pharm. 2005, 62(2): 173-181). The in
vivo concentration of Exenatide after a single subcutaneous injection is
increased persistently and can arrive to the maximum plasma concentration
after 2 h or so, which can be maintained for 4-6 hours. (See Nielsen L L,
Baron A D. Pharmacology of exenatide (synthetic exendin-4) for the
treatment of type 2 diabetes. Curr Opin Investig Drugs. 2003,
4(4):401-05). It should be noted that the metabolism of Exenatide does
not occur in the liver, but is degraded mainly by protein protease after
filtered by renal glomeruli.

[0018] Exenatide has special glucose-regulating activities, including
glucose-dependent enhance of insulin secretion, glucose-dependent
inhibition of wrong excessive glucagon secretion, slowing gastric
emptying and decreasing food intake and the like. Studies in vitro and in
vivo in the models of diabetes found that Exenatide also has the effects
of storing the first stage (first-phase) insulin secretion, promoting the
proliferation of β-cell and promoting the regeneration of insulin
from its precursor cell.

[0019] In order to achieve better control of blood glucose, injections
twice a day of Exenatide are needed. This is a major inconvenience to
patients. Furthermore, Exenatide has unfortunate side effects including
mild to moderate nausea (about 40% of patients will have this reaction),
diarrhea and vomiting (less than 15% of patients have both reactions). In
addition, about 50% of Exenatide-treated patients can generate
antibodies, although these antibodies do not affect the efficacy or lead
to other clinical effects. Recently it is found that six patients
suffered hemorrhage or symptoms of necrotizing pancreatitis after taking
Byetta®.

[0020] CJC-1131 is a GLP-1 analogue with peptidase resistance developed by
ConjuChem Biotechnologies Inc., in which the alanine residue in the
second position of GLP-1 is replaced with D-Ala to enhance resistance of
DPP-IV enzymolysis. The structure contains an active reactive linker that
can bind to serum albuminutesthrough a covalent, non-reversible manner.
(See Kim J G, Baggio L L, Bridon D P, et al. Development and
characterization of a glucagon-like peptide-1 albuminutesconjugate: the
ability to activate the glucagon-like peptide 1 receptor in vivo.
Diabetes 2003, 52(3):751-759). The GLP-1-serum albuminutescomplex retains
the activity of GLP-1, while increasing its stability to DPP-IV
enzymolysis, thereby extending in vivo action. Its half-life in plasma is
about 20 days.

[0021] A study has found that the Ki was approximate 12 nM (the Ki of
GLP-1 is 5.2 nM) when CJC-1131-serum albuminutescomplex is bound to
Chinese hamster ovary cell transfected with recombinant human pancreatic
GLP-1 receptor. Meanwhile the EC50 of the complex activating cAMP is
11-13 nM, wherein the EC50 is similar to GLP-1's EC50. Existing
literature reports show that this complex can reduce postprandial blood
glucose level of the mice whose blood sugar is normal or high, and tests
show that this activity of CJC-1131 acts on a certain functional receptor
of GLP-1. Meanwhile in mice, CJC-1131 also has an effect on slowing
gastric emptying and inhibiting food intake and the like.

[0022] Part of a phase II clinical trial of CJC-1131 has been completed.
In September 2005, ConjuChem concluded that CJC-1131 may not be suitable
for chronic dosing regimens after analysis of test results and suspended
further clinical study

[0023] Albugon (albumin-GLP-1) is a long-acting drug for the treatment of
T2DM developed by GlaxoSmithKline authorized by Human Genome Sciences
Inc., which is a fusion protein of GLP-1 (with mutations increasing the
resistance to DDP-IV) and albumin. Its half-life in monkeys is 3 days.
The basic idea of the development thereof is to couple the recombinant
GLP-1 and serum albuminutes to form a complex, thereby its in vivo
half-life is significantly increased. The administration of Albugon
effectively reduces blood glucose level of mice, increases insulin
secretion, slows gastric emptying and reduces food intake etc. (See
Baggio L L, Huang Q, Brown T J, et al. A Recombinant Human Glucagon-Like
Peptide (GLP)-1-Albuminutes Protein (Albugon) Mimics Peptidergic
Activation of GLP-1Receptor-Dependent Pathways Coupled With Satiety,
Gastrointestinal Motility, and Glucose Homeostasis. Diabetes 2004,
53(9):2492-2500). Currently Albugon is in phase III clinical trials.

[0024] WO9808871 discloses a GLP-1 derivative which is obtained through
the modification on GLP-1 (7-37) with fatty acid. The half life in vivo
of GLP-1 is significantly enhanced. WO9943705 discloses a derivative of
GLP-1, which is chemically modified at the N-terminus, but some
literature reports that modification of the amino acids on the N-terminal
will significantly decrease the activity of the entire GLP-1 derivative.
(See J. Med. Chem. 2000, 43, 1664 1669). In addition, CN200680006362,
CN200680006474, WO2007113205, CN200480004658, CN200810152147 and
WO2006097538 etc also disclose a series of GLP-1 analogues or derivatives
thereof produced by chemical modification or amino acid substitution, in
which the most representative one is liraglutide developed by Novo
Nordisk, the phase III clinical trial of which has been finished.
Liraglutide is a derivative of GLP-1, whose structure contains a GLP-1
analogue of which the sequence is 97% homologous with human GLP-1, and
this GLP-1 analogue is linked with palmitic acid covalently to form
Liraglutide, wherein the palmitic acid of the structure of Liraglutide is
linked to serum albuminutes non-covalently, and this structural
characteristic affects a slower release from the injection site without
changing the activity of GLP-1 thereby extending its in vivo half life
Meanwhile, the palmitic acid in the structure will form a certain steric
hindrance to prevent the degradation by DPP-IV and to reduce renal
clearance. Because of the characteristics described above, the half-life
of Liraglutide in the human body administered by subcutaneous injection
is about 10-14 hours. In theory, it can be administered once on day and
the daily dose is 0.6-1.8 mg. On Apr. 23, 2009, Novo Nordisk announced
that Committee for Medicinal products for Human Use (CHMP) under the EMEA
gave a positive evaluation on Liraglutide and recommended approval of its
listing. Novo Nordisk hopes that European Commission would approve its
application of listing within two months.

BRIEF SUMMARY OF THE INVENTION

[0025] The present invention describes GLP-1 analogues which have longer
half-life in vivo. The GLP-1 analogues described have the same function
as that of human GLP-1 and a longer half-life in vivo.

[0026] The present invention also includes pharmaceutical compositions
comprising GLP-1 analogues and pharmaceutically acceptable salts thereof,
for use in the treatment of non-insulin-dependent diabetes mellitus,
insulin-dependent diabetes and obesity.

[0027] The aims of the present invention are achieved by the following
technical solutions. The present invention provides GLP-1 analogues
having amino acid sequence of formula (I) or a pharmaceutically
acceptable salt thereof:

[0028] The GLP-1 analogues refer to a new GLP-1 peptide obtained by the
partial amino acid substitution or the extension at the C terminal of
human GLP-1 (7-37) peptide serving as a precursor, comprising GLP-1
(7-36) amide and GLP-1 (7-37), which has same function as that of human
GLP-1.

[0029] The GLP-1 analogues may be modified so that amino acid residues
have lipophilic substituents, wherein a typical modification is to form
an amide or ester, preferably, to form an amide.

[0030] In a preferred embodiment of the invention, the lipophilic
substituent of formula R1(CH2)n--CO-- and the amino group
of the amino acid residues of the GLP-1 analogue are linked by an amide
bond, in which R1 is selected from CH3-- and HOOC--, and n is
an integer selected from 8-25.

[0031] In another preferred embodiment of the invention, the lipophilic
substituent of formula R1(CH2)n--CO-- and the ε
amino group of the Lys at the C-terminal of the GLP-1 analogue are linked
by an amide bond, in which R1 is selected from CH3-- and
HOOC--, and n is an integer selected from 8-25.

[0032] In yet another preferred embodiment of the invention, the
lipophilic substituent of formula RI(CH2)n--CO-- and the
α amino group of the Lys at the C-terminal of the GLP-1 analogue
are linked by an amide bond, in which R1 is selected from CH3--
and HOOC--, and n is an integer selected from 8-25, and 14 is the most
preferred.

[0033] In another preferred embodiment of the invention, X1 in the amino
acid sequence of the GLP-1 analogue is selected from L-His and D-His; X2
is selected from Ala, D-Ala, Gly, Val, Leu, Ile, Lys and Aib; X10 is
selected from Val and Leu; X12 is selected from Ser, Lys and Arg; X13 is
selected from Tyr and Gln; X14 is selected from Leu and Met; X16 is
selected from Gly, Glu and Aib; X17 is selected from Gln, Glu, Lys and
Arg; X19 is selected from Ala and Val; X20 is selected from Lys, Glu and
Arg; X21 is selected from Glu and Leu; X24 is selected from Val and Lys;
X27 is selected from Val and Lys; X28 is selected from Lys, Glu, Asn and
Arg; X29 is selected from Gly and Aib; X30 is selected from Arg, Gly and
Lys; X31 is selected from Gly, Ala, Glu, Pro and Lys; X32 is selected
from Lys and Ser; X33 is selected from Lys and Ser; X34 is selected from
Gly, Ala and Sar; X35 is selected from Gly, Ala and Sar; X36 is selected
from Pro and Gly; X37 is selected from Pro and Gly; X38 is selected from
Pro and Gly; X39 is selected from Ser and Tyr.

[0034] In one more preferred embodiment of the present invention, the
amino acid sequence of the GLP-1 analogue is selected from the group
consisting of SEQ ID NO: 1 to SEQ ID NO: 120.

[0035] In another preferred embodiment of the present invention, the
lipophilic substituent of formula R1(CH2)n--CO-- and the
amino group of the amino acid residues of the GLP-1 analog, of which the
sequence is selected from the group consisting of SEQ ID NO: 1 to SEQ ID
NO: 120, are linked by an amide bond, in which R1 is selected from
CH3-- and HOOC--, and n is an integer selected from 8-25.

[0036] In one more preferred embodiment of the present invention, the
lipophilic substituent of formula R1(CH2)n--CO-- and the
ε amino group of the C-terminal Lys of the GLP-1 analog, selected
from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 120, are linked
by an amide bond, in which R1 is selected from CH3-- and
HOOC--, and n is an integer selected from 8-25.

[0037] In one more preferred embodiment of the present invention, the
lipophilic substituent of formula R1(CH2)n--CO-- and the
α amino group of the C-terminal Lys of the GLP-1 analog, selected
from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 120, are linked
by an amide bond, in which R1 is selected from CH3 and HOOC--,
and n is an integer selected from 8-25, preferably n is selected from 8,
10, 12, 14, 16, 18, 20 and 22, most preferably, n is 14.

[0038] In one more preferred embodiment of this invention, the lipophilic
substituent of formula R1(CH2)n--CO-- and the α
amino group of the C-terminal Lys of the GLP-1 analog, selected from the
group consisting of SEQ ID NO: 1 to SEQ ID NO: 20, are linked by an amide
bond, in which R1 is selected from CH3-- and HOOC--, and n is
an integer selected from 8-25, preferably n is selected from 8, 10, 12,
14, 16, 18, 20 and 22, most preferably, n is 14.

[0039] In another more preferred embodiment of this invention, the
lipophilic substituent of formula R1(CH2)n--CO-- and the
α amido of the C-terminal Lys of the GLP-1 analog, selected from
the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8, are linked by an
amide bond, in which R1 is CH3, and n is 14.

[0041] The pharmaceutical compositions containing GLP-1 derivatives
according to the invention can be used to treat patients who need this
treatment by the way of parenteral administration. Parenteral
administration can be chosed from subcutaneous, intramuscular or
intravenous injections. The GLP-1 derivatives of the invention can also
be administered by transdermal routes, such as administration via
transdermal patch (iontophoresis patch and others) and administration
through the mucosa.

[0042] The pharmaceutical compositions containing the GLP-1 derivatives of
the invention can be prepared through common techniques in the art of
pharmaceutical industry. These techniques comprise proper dissolving and
mixing the components to obtain the desired final compositions. For
instance, the GLP-1 derivatives are dissolved in a certain amount of
water, wherein the volume of water is slightly less than the final volume
of the obtained composition. Isotonic agents, preservatives, surfactants
and buffers are added according to need, wherein said isotonic agents are
sodium chloride, mannitol, glycerol, propylene glycol, sugar or alditol.

[0043] Said preservatives are phenol, orthocresol, para-cresol,
meta-cresol, methylparahydroxybenzoate ester, benzyl alcohol. Said
appropriate buffering agents are sodium acetate, sodium carbonate,
glycine, histidine, lysine, sodium dihydrogen phosphate, disodium
hydrogen phosphate, sodium phosphate. Said surfactants are Poloxamer,
Poloxamer-188, Poloxamer-407, Tween 80 and Tween-20. If necessary, the
aqueous solutions of acids such as hydrochloric acid or alkali such as
sodium hydroxide solution are added to adjust pH values of the solutions,
and finally the solution volume is adjusted by adding water to obtain the
required concentration. Besides said components, the pharmaceutical
compositions of the invention also comprise enough basic amino acids or
other alkaline reagents having the function to decrease the aggregates
formed by the composition during storage, such as lysine, histidine,
arginine, imidazole during storage.

[0044] The GLP-1 analogues of the invention can be synthesized manually,
wherein the resin is HMPA-AM resin, the α-amino group of the amino
acid derivatives is protected by the Fmoc (fluorene formyl carbonyl), the
side-chain thiol of cysteine, the side-chain amido of glutamine, the
side-chain imidazole of histidine are protected by Trt (triphenylmethyl),
the side-chain guanidyl of arginine is protected by Pbf
(2,2,4,6,7-pentamethyl-dihydrobenzo furan-5-sulfonyl). The side-chain
indolyl of tryptophan and the side-chain amino group of lysine are
protected by Boc (tert-Butoxycarbonyl) (the side-chain amino group of the
Lys are protected by Mtt when the peptide backbone is formed through
ε amino group of Lys), the side-chain hydroxyl of threonine, the
side-chain phenylol of tyrosine, the side-chain hydroxyl of serine are
protected by tBu (tert-butyl). The carboxyl of C-terminal amino acids of
the peptide chain of the GLP-1 analogues which will be synthesized is
connected with an insoluble high molecular resin (HMP-AM resin) through
covalent bonds, and then, the amino acids bound to a solid phase carrier
act as amino components, the amino protection group is removed by 20%
Hexahydropyridine/DMF solution, and then reactes with excess amino acid
derivatives to link a long peptide chain. The operation
(Condensation→washing→deprotection→washing→ne-
xt round of condensation) is repeated to achieve the peptide chain length
desired. Finally, the peptide chain is cleaved down from the resins by
using mixture of TFA: water: 1,2-dithioglycol:
triisopropylsilane(92.5:2.5:2.5:2.5), to obtain the crude GLP-1 analogues
through precipitation in an ether. The crude products are purified
through using C18 reversed-phase column, and thereby obtaining the
desired GLP-1 analogues. The ninhydrin testing method was used to moniter
the condensation and the deprotection steps--that is, when there are free
aminos on the resin, the ninhydrin reagent will show blue and no color
(or slightly yellow) will be shown when there are no free aminos on the
resin (Ninhydrin reagent itself is yellow). Therefore, after the
condensation reaction is completed, if it shows yellow through ninhydrin
test (color of Ninhydrin reagent per se), then it suggests that the
coupling step is completed and the deprotection operation before next
amino acid coupling can be carried out. If the testing shows blue, it
suggests that there are still some free aminos on the peptide chains, and
it is needed to further repeat the coupling step or to change the
existing condensing agent until the testing has no color or slightly
yellow.

DETAILED DESCRIPTION OF THE INVENTION

[0045] To describe the present invention in more detail, the following
examples are provided. However, the present invention should not be
construed as limited to the embodiments set forth herein.

Example 1

The Method for Solid-Phase Synthesis of HS-20001

1. Preparation of Fmoc-Lys (Mtt)-HMP-AM Resin

(1) Drying and Swelling of HMP-AM Resin

[0046] 50 g (30 mmol) HMP-AM resin (0.6 mmol/g) was dried for 24 hours in
vacuum and placed into a 2 L bubbling bottle. Resins were swelled with
500 mL N,N-dimethylformamide (DMF) for 30 minutes, then the DMF was
drawn-off and the resins were washed with DMF for 1 minute. The washing
step was repeated twice.

2. Preparation of Fmoc-Lys(Mtt)-HMP-AM Resin

[0047] (1) Coupling of Fmoc-Lys (Mtt)-OH with HMP-AM Resin

[0048] The resins were washed with 500 mL DCM and then the washing step
was repeated twice. 56.2 g (90 mmol) Fmoc-Lys(Mtt)-OH and 11.4 g (90
mmol) DIC were dissolved in 1 L DCM and added into the swelled HMP-AM
resin. 366 mg (3 mmol) DMAP were added to react for 24 hours.

(2) Washing of the Resin

[0049] After the reaction, the resin was washed alternately with DMF and
IPA twice and washed with DMF 3 times.

[0051] The resin was washed twice with 1 L 50% MeOH/DMF, 50% DCM/DMF, and
then washed three times with DCM and with dehydrated ethanol three times
successively. The resin was then dried under vacuum to obtain the
Fmoc-Lys(Mtt)-HMP-AM resin.

[0055] The resin was washed with 200 mL DCM twice followed by addition of
1200 mL 1% TFA/DCM (TFA is about 8-fold excess) to remove Mtt protecting
group for 1 hour. The resin was alternately washed with 200 mL 5%
N,N-diisopropyl ethylamine (DIEA)/DMF and DMF three times followed by DMF
washing three times.

4. Palmitic Acid Condensation 50 mmol palmitic acid and 50 mmol
3-(diethoxyphosphoryloxy)-1,2,3-phentriazine-4-ketone (DEPBT) were
dissolved in 400 mL DMF. Then 100 mmol DIEA were added and stirred for 3
minutes at room temperature. The solution was added to the resin, reacted
in 37° C. water baths for 2 hours under N2. After the
reaction, the reaction solution was drawn-off and the resin was washed
with DMF, isopropyl alcohol (IPA), and DMF in turn. 5. \Removal of 9-Fmoc
(Fluorenylmethyloxycarbonyl) Protecting Group of Fmoc-Lys
(N-ε-palmitic acid)-HMPA-AM Resin

[0056] 200 mL 20% piperidine/DMF solution were placed into a bubbling
bottle filled with Fmoc-Lys (N-ε-palmitic acid)-HMPA-AM resin and
reacted for 5 minutes and then is drawn out. Then 200 mL 20%
piperidine/DMF solution were added to react for 20 minutes at room
temperature. After the reaction, the resin was washed with 200 mL DMF
four times.

6. Solid Phase Synthesis of Peptide Chain Part of HS-20001

[0057] (1) Condensation of Fmoc-Ser (tBu)-OH

[0058] 50 mmol Fmoc-Ser(tBu)-OH were dissolved in 125 mL 0.4 M
1-hydroxybenzo triazole (HOBt)/DMF. Then 125 mL 0.4 M N,N'-diisopropyl
carbodiimide (DIC)/DCM were added to activate and react for 10 minutes at
room temperature. The solution was added into the resin, reacted under of
nitrogen at room temperature. Ninhydrin was used to detect and control
the degree of the reaction. After reaction, the reaction solution was
removed, and the resin was washed with DMF, IPA and DMF in turn.

(2) Extension of the Peptide Chain

[0059] HS-20001 resin peptide was synthesized according to the sequence of
the peptide chain of HS-20001 from the amino terminal (N-terminal) to the
carboxy-terminal (C-terminal)
(His-(D)-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Nle-Glu-Glu-Glu--
Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Gln-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pr-
o-Pro-Pro-Ser), wherein the amounts of amino acids and condensation
reagents were the same as the amounts for Fmoc-Ser (tBu)-OH. Protected
amino acids were Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Val-OH, Fmoc-Nle-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH,
Fmoc-D-Ala-OH and Fmoc-His(Trt)-OH respectively, and condensation and
deprotection reactions were repeated.

(3) Post-Processing of HS-20001 Resin Peptide

[0060] HS-20001 resin peptide obtained in step (2) was washed with DMF,
IPA and DMF in turn, then washed with absolute ether twice, and dried
under vacuum to obtain the HS-20001 resin peptide.

(4) Preparation of HS-20001 Crude Peptide

[0061] The dried HS-20001 peptide resin is reacted with fresh lysate), of
trifluoroacetic acid (TFA): triisopropylsilane (TIS): water=95:2.5:2.5
(by volume and total 10 mL of lysate per gram of the dry resin) for 4 h
at room temperature. The reaction solution was filtrated, and the resin
was washed with TFA twice. The filtrate was collected, combined, and
concentrated to 1/3 of the original volume through rotary evaporation.
HS-20001 was precipitated and washed with cold absolute ether, after
centrifugation and drying in vacuum, white crude HS-20001 was obtained.

the column is Denali C-18 column (particle diameter 8.3 μm, 5×30
cm), column temperature is 45° C., detection wavelength is 220 nm,
flow rate is 120 mL/min. The product peaks were collected and
concentrated under vacuum to remove most of the acetonitrile. 2.25 g of
the product (HS-20001) was obtained by lyophilization, of which the
purity as 98.5%, and the yield was 22.5%.

Example 2

The solid-phase synthesis method for HS-20002

1. Preparation of Fmoc-Lys (Mtt)-HMP-AM Resin

[0063] See Example 1.

2. Swelling of the Solid-Phase Synthesized Resin

[0064] 50 g (20 mmol) Fmoc-Lys(Mtt)-HMPA-AM resin (0.4 mmol/g) was dried
for 24 hours in vacuum and placed into a 2 L bubbling bottle. The resin
was swelled with 500 mL DMF for 30 minutes, and then DMF solution was
drawn-off

3. Removal of Mtt Protecting Group of Fmoc-Lys(Mtt)-HMPA-AM Resin

[0065] The resin was washed with 200 mL DCM twice. Mtt protecting group
was removed by adding 1200 mL 1% TFA/DCM (TFA is about 8-fold excess) for
1 hour, and then washed with 200 mL 5% DIEA/DMF and DMF alternately for
three times followed by DCM washing three times.

4. Palmitic Acid Condensation

[0066] 50 mmol palmitic acid and 50 mmol DEPBT were dissolved in 400 mL
DMF, and then 100 mmol DIEA was added by stirring to react for 3 minutes
at room temperature. The resulting solution was added to the resin and
reacted in 37° C. water bath under N2 for 2 hours. After the
reaction, the reaction solution as removed, and the resin was washed with
DMF, isopropyl alcohol (IPA), and DMF in turn.

[0067] 200 mL 20% Piperidine/DMF solution was placed into a bubbling
bottle filled with Fmoc-Lys(N-ε-palmitic acid)-HMPA-AM resin, and
drawn-off after reacting for 5 minutes. 200 mL 20% Piperidine/DMF
solution was added for reacting for 20 minutes at room temperature. After
the completion, the resin was washed four times with 200 mL DMF.

6. Solid-Phase Synthesis Method for the Peptide Chain Part of HS-20002

[0068] (1) Condensation of Fmoc-Ser (tBu)-OH

[0069] 50 mmol Fmoc-Ser(tBu)-OH were dissolved in 125 mL 0.4M HOBt/DMF,
then 125 mL 0.4 M DIC/DCM were added to activate and react for 10 minutes
at room temperature. The resulting solution was contacted with the resin
and reacted under N2 at room temperature. Ninhydrin was used to
detect and control the degree of the reaction. After the reaction, the
reaction solution as removed, and the resin was washed with DMF, IPA and
DMF in turn.

(2) Extension of the Peptide Chain

[0070] HS-20002 resin peptide was synthesized according to the sequence of
peptide chain of HS-20002 from the N-amino (N-terminal) to the
carboxy-terminal (C-terminal)
(His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala--
Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pr-
o-Pro-Ser), wherein the amounts of amino acids and condensation reagents
were the same as that of Fmoc-Ser (tBu)-OH, protected amino acids were
Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asp(OtBu)-OH,
Fmoc-Thr(tBu)-OH and Fmoc-His(Trt)-OH respectively, and condensation and
deprotection reactions were repeated.

(3) Post-Processing of HS-20002 Resin Peptide

[0071] The HS-20002 resin peptide obtained in step (2) was washed with
DMF, IPA and DMF in turn, then washed twice with absolute ether, then
dried under vacuum. HS-20002 resin peptide was obtained therefrom.

(4) Preparation of HS-20002 Crude Peptide

[0072] The dried HS-20002 peptide resin was reacted with fresh lysate of
trifluoroacetic acid (TFA): triisopropylsilane (TIS): water:
1,2-ethanedithiol (EDT)=94:1:2.5:2.5 (by volume and total 10 mL of lysate
per gram of the dry resin) for 4 hours at room temperature. The reaction
solution was filtrated after the reaction. The resin was washed with TFA
twice, and then filtrate was collected, combined, and concentrated to 1/3
of the original volume through rotary evaporation. HS-20002 was
precipitated with cold absolute ether, after centrifugation and drying
under vacuum. The resulting product was white crude HS-20002.

(5) Preparation of HS-20002 with Reversed-Phase Liquid Chromatography

[0073] 10 g crude HS-20002 were dissolved in a certain amount of water,
filtrated with 0.45 μm membrane filter, then purified with
reversed-phase high performance liquid chromatography (RP-HPLC), with a
mobile phase A was 0.1% TFA/H2O, B 0.1% TFA/acetonitrile, the column
was a Denali C-18 column (particle diameter 8.3 μm, 5×30 cm),
column temperature was 45° C., detection wavelength was 220 nm,
flow rate was 120 mL/min. The product peaks were collected, concentrated
under vacuum to remove most of acetonitrile. 2.1 g of HS-20002 was
obtained by lyophilization, of which the purity was 98%, and the yield
was 20.5%.

Example 3

The Solid-Phase Synthesis Method for HS-20003

1. Preparation of Fmoc-Lys (Mtt)-HMP-AM Resin

[0074] See Example 1.

2. Swelling of the Solid-Phase Synthesized Resin

[0075] 50 g (20 mmol) Fmoc-Lys(Mtt)-HMPA-AM resin (0.4 mmol/g) dried for
24 hours in vacuum were placed into a 2 L bubbling bottle. The resin was
swelled with 500 mL DMF for 30 minutes, and then DMF solution was
drawn-off

3. Removal of Fmoc Protecting Group of Fmoc-Lys(Mtt)-HMPA-AM Resin

[0076] 200 mL 20% piperidine/DMF solution were added into a bubbling
bottle filled with Fmoc-Lys (Mtt)-HMPA-AM resin. Then the solution was
drawn off after 5 minutes, and 200 mL 20% piperidine/DMF solution were
added. The reaction continued for another 20 minutes at room temperature.
After the reaction, the resin was washed four times with 200 mL DMF.

4. Palmitic Acid Condensation

[0077] 50 mmol Palmitic acid and 50 mmol DEPBT were dissolved in 400 mL
DMF. Then 100 mmol DIEA was added by stirring to react for 3 minutes at
room temperature. The resulting solution was added to the resin, reacted
in 37° C. water baths under N2 for 2 hours. After the
reaction, the reaction solution was removed, and the resin was washed
with DMF, isopropyl alcohol (IPA), and DMF in turn.

5. Removal of Mtt Protecting Group of N-ε-Palmitic
acid-Lys(Mtt)-HMPA-AM Resin

[0078] The resin was washed with 200 mL DCM twice. The Mtt protecting
group was removed by adding 1200 mL 1% TFA/DCM (TFA is about 8-fold
excess) for 1 hour. The resin was washed with 200 mL 5% DIEA/DMF and DMF
alternately three times, then washed with DCM three times.

6. Solid-Phase Synthesis Method for the Peptide Chain Part of HS-20003

[0079] (1) Condensation of Fmoc-Ser (tBu)-OH

[0080] 50 mmol Fmoc-Ser(tBu)-OH and 50 mmol DEPBT were dissolved in a
certain amount of DCM. Then 100 mmol DIEA was added for activation for 3
minutes at room temperature. The solution was added to the resin, reacted
under N2 at room temperature, and ninhydrin was used to detect and
control the degree of the reaction. After the reaction, the reaction
solution was removed, and the resin was washed with DMF, IPA and DMF in
turn.

(2) Extension of the Peptide Chain

[0081] HS-20003 resin peptide is synthesized according to the sequence of
peptide chain of HS-20003 from the N-amino (N-terminal) to the
carboxy-terminal (C-terminal)
(His-(D)-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Glu--
Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pr-
o-Pro-Pro-Ser), wherein the amounts of amino acids and condensation
reagents were the same as that of Fmoc-Ser (tBu)-OH, protected amino
acids were Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH respectively, and
condensation and deprotection reactions were repeated.

(3) Post-Processing of HS-20003 Resin Peptide

[0082] The HS-20003 resin peptide obtained in step (2) was washed with
DMF, IPA and DMF in turn, then washed twice with absolute ether, and
dried under vacuum to obtain HS-20003 resin peptide.

(4) Preparation of HS-20003 Crude Peptide

[0083] The dried HS-20003 peptide resin was reacted with fresh lysate of
trifluoroacetic acid (TFA): triisopropylsilane (TIS): water=95:2.5:2.5
(by volume and total 10 mL of lysate per gram of the dry resin) for 4
hours at room temperature. The reaction solution was filtrated after the
reaction. The resin was twice washed with TFA. The filtrate was
collected, combined, and concentrated to 1/3 of the original volume
through rotary evaporation. HS-20003 was precipitated with cold ether
under stirring. After centrifugation and drying in vacuum, white crude
HS-20003 was obtained.

(5) Preparation of HS-20003 with Reversed-Phase Liquid Chromatography

[0084] 10 g crude HS-20003 was dissolved in a certain amount of 20% acetic
acid/water and stirred for at least 4 hours, then filtrated with 0.45
μm membrane filter, then purified with reversed-phase high performance
liquid chromatography (RP-HPLC), wherein the mobile phase was A 0.1%
TFA/H2O, B 0.1% TFA/acetonitrile, the column was Denali C-18 column
(particle diameter 8.3 μm, 5×30 cm), column temperature was
45° C., detection wavelength was 220 nm, flow rate was 120 mL/min.
The product peaks were collected, concentrated with vacuum to remove most
of acetonitrile. 2.5 g of HS-20003 was obtained by lyophilization, of
which the purity was 98.5%, and the yield was 25%.

[0087] 200 mL 20% piperidine/DMF solution were added into a bubbling
bottle filled with Fmoc-Lys (Mtt)-HMPA-AM resin, and then drawn off after
5 minutes, and then 200 mL 20% piperidine/DMF solution was added for
reacting for 20 minutes at room temperature. After the reaction, the
resin was washed four times with 200 mL DMF.

4. Palmitic Acid Condensation

[0088] 50 mmol palmitic acid and 50 mmol DEPBT were dissolved in 400 mL
DMF, and then 100 mmol DIEA was added by stirring for 3 minutes at room
temperature. The resulting solution was added to the resin, reacted in
37° C. water bath under N2 for 2 hours. After the reaction,
the reaction solution was removed, and the resin was washed with DMF,
isopropyl alcohol (IPA) and DMF in turn.

5. Removal of Mtt Protecting Group of Palmitic Acid-Lys(Mtt)-HMPA-AM Resin

[0089] The resin was washed with 200 mL DCM twice. Mtt protecting group
was removed by adding 1200 mL 1% TFA/DCM (TFA is about 8-fold excess) for
reacting for 1 hour, then washed with 5% DIEA/DMF and DMF alternately for
three times, then washed three time with DCM.

6. The Solid-Phase Synthesis Method for the Peptide Chain Part of HS-20004

[0090] (1) Condensation of Fmoc-Ser (tBu)-OH

[0091] 50 mmol Fmoc-Ser(tBu)-OH and 50 mmol DEPBT were dissolved in a
certain amount of DCM. Then 100 mmol DIEA was added for activation for 3
minutes at room temperature. The resulting solution was added to the
resin, reacted under N2 at room temperature, and ninhydrin was used
to detect and control the degrees of the reaction. After the reaction,
the reaction solution was removed, and the resin was washed with DMF, IPA
and DMF in turn.

(2) Extension of the Peptide Chain

[0092] HS-20004 resin peptide was synthesized according to the sequence of
the peptide chain of HS-20004 from the N-amino (N-terminal) to the
carboxy-terminal (C-terminal)
(His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Glu-Ala--
Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pr-
o-Pro-Ser), wherein the amounts of amino acids and condensation reagents
were same as that of Fmoc-Ser (tBu)-OH. Protected amino acids were
Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Aib-OH and
Fmoc-His(Trt)-OH respectively, and the condensation and deprotection
reactions were repeated.

(3) Post-Processing for HS-20004 Resin Peptide

[0093] HS-20004 resin peptide obtained in step (2) was washed with DMF,
IPA and DMF in turn, then washed twice with absolute ether, followed by
drying under vacuum to obtain HS-20004 resin peptide.

(4) Preparation of Crude HS-20004 Peptide

[0094] The dried HS-20004 resin peptide was reacted with fresh lysate of
trifluoroacetic acid (TFA): triisopropylsilane (TIS): water=95:2.5:2.5
(by volume and total 10 mL of lysate per gram of the dry resin) for 4
hours at room temperature. The reaction solution was filtrated, and the
resin was washed twice with TFA. The filtrate was collected, combined,
and concentrated to 1/3 of the original volume through rotary
evaporation. HS-20004 was precipitated with cold ether under stirring.
After centrifugation and drying in vacuum, white crude HS-20004 was
obtained.

(5) Preparation of HS-20004 with Reversed-Phase Liquid Chromatography

[0095] 10 g crude HS-20002 was dissolved in a certain amount of 20% acetic
acid/water and stirred for at least 4 hours, then filtrated with 0.45
μm membrane filter, and purified with reversed-phase high performance
liquid chromatography (RP-HPLC), wherein mobile phase was A 0.1%
TFA/H2O, B 0.1% TFA/acetonitrile, the column was Denali C-18 column
(particle diameter 8.3 μm, 5×30 cm), column temperature was
45° C., detection wavelength was 220 nm, flow rate was 120 mL/min.
The product peaks were collected, concentrated with under vacuum to
remove most of acetonitrile. 2.25 g of HS-20004 was obtained by
lyophilization, of which the purity was 98.5%, and the yield was 22.5%.

Example 5

The Solid-Phase Synthesis Method for HS-20005

[0096] The preparation method of HS-20005 is as same as that described in
example 4, wherein the difference is that the amino acid sequence is
replaced with SEQ ID NO: 5, and 2.5 g HS-20005 product was obtained, the
purity of which was 98.5%, and the yield was 25%.

Example 6

The Solid-Phase Synthesis Method for HS-20006

[0097] The preparation method of HS-20006 was the same as that described
in example 4, wherein the difference was that the amino acid sequence was
replaced with SEQ ID NO: 6. 2.25 g of HS-20006 product was obtained, the
purity of which is 98.5%, and the yield is 22.5%.

Example 7

The Solid-Phase Synthesis Method for HS-20007

[0098] The preparation method of HS-20007 was the same as that described
in example 4, wherein the difference is that the amino acid sequence was
replaced with SEQ ID NO: 7. 2.1 g of HS-20007 product was obtained, the
purity of which was 98%, and the yield was 20.5%.

Example 8

The Solid-Phase Synthesis Method for HS-20008

[0099] The preparation method of HS-20008 was the same as that described
in example 4, wherein the difference is that the amino acid sequence was
replaced with SEQ ID NO: 8. 2.5 g of HS-20008 product was obtained, the
purity of which was 98.5%, and the yield was 25%.

Reference Example Solid-Phase Synthesis Method for Liraglutide

1. Preparation of Fmoc-Lys(Mtt)-HMP-AM Resin

[0100] (1) Drying and swelling of HMP-AM resin

[0101] 50 g (30 mmol) HMP-AM resin (0.6 mmol/g) was dried for 24 hours in
vacuum and placed into a 2 L bubbling bottle. 500 mL
N,N-dimethylformamide (DMF) was added to swell therein for 30 minutes.
The DMF solution was drawn-off, and DMF was added to wash the resin for 1
minute. This washing step was repeated twice.

[0106] The resin was washed twice with 1 L 50% MeOH/DMF, 50% DCM/DMF,
three times with DCM, and was washed three times with absolute ethanol.
It was then dried under vacuum to obtain the Fmoc-Lys(Mtt)-HMP-AM resin.

(3) Loading assays of Fmoc-Lys(Mtt)-HMP-AM Resin 5˜10 mg resin were
put into 1 mL 20% Hexahydropyridine/DMF solution and stirred for 20
minutes. 50 μL supernatant is taken with a pipet and diluted in 2.5 mL
DMF. Blank samples: 50 μL 20% Hexahydropyridine/DMF was taken with a
pipet and is diluted in 2.5 mL DMF. Degree of substitution is calculated
as follows:

Sub=(A×51)/(7.8×m)

wherein A is the absorption value of UV at 301 nm; m is the weight of the
resin in mg.

3. The Solid Phase Synthesis Method of the Peptide Chain Part of
Liraglutide

[0108] {circle around (1)} condensation of Fmoc-Arg(Pbf)-OH

[0109] 50 mmol Fmoc-Arg(Pbf)-OH were dissolved in 125 mL 0.4M
1-hydroxybenzotriazole (HOBt)/DMF, then 125 mL 0.4M
N,N'-diisopropylcarbodiimide (DIC)/DCM were added to activate and react
for 10 minutes at room temperature. The resulting solution was added to
the resin, reacted under N2 at room temperature, and ninhydrin was
used to detect and control the degrees of the reaction. After the
reaction, the reaction solution was drawn off, and the resin was washed
with DMF, IPA and DMF in turn.

{circle around (2)} Extension of the Peptide Chain

[0110] Precursor peptide of Liraglutide was synthesized according to the
sequence of the peptide chain of Liraglutide from the N-amido
(N-terminal) to the carboxy-terminal (C-terminal)
(His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala--
Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly), wherein the amounts
of amino acids and condensation reagents were the same as that of
Fmoc-Arg(Pbf)-OH, protected amino acids were Fmoc-Arg(Pbf)-OH,
Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH,
Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH,
Fmoc-His(Trt)-OH respectively, and the condensation and deprotection
reactions were repeated.

{circle around (3)} Removal of Mtt Protecting Group of the Precursor
Peptide of Liraglutide

[0111] The resin was twice washed with 200 mL DCM. The Mtt protecting
group was removed twice by adding 1200 mL 1% TFA/DCM (TFA is about 8-fold
excess) to react for 1 hour. The resin was washed alternately with 200 mL
5% N,N-diisopropylethylamine (DIEA)/DMF and DMF for three times, and
washed 3 times with DMF.

{circle around (4)} Modification of Precursor Peptide of Liraglutide with
Palmitic Acid

[0112] 50 mmol Fmoc-Glu-OtBu were dissolved in 125 mL 0.4 M 1-hydroxybenzo
triazole (HOBt)/DMF. Then 125 mL 0.4M N,N'-diisopropylcarbodiimide
(DIC)/DCM was added to activate and react for 10 minutes at room
temperature. The solution was added to the resin from step {circle around
(3)}, and allowed to react under N2 at room temperature.

[0113] Ninhydrin was used to detect and control the degree of the reaction
or reaction progress. After the reaction, the reaction solution was
drawn-off, and the resin is washed with DMF, IPA and DMF in turn.

[0114] 1 L 20% PIP/DMF was added to remove Fmoc protecting group for 5
minutes, then drawn off 1 L of 20% PIP/DMF was added to remove Fmoc
protecting group for 20 minutes, then are drawn off The resulting resin
was washed four times with DMF.

[0115] 50 mmol palmitic acid and 50 mmol
3-(diethoxyphosphoryloxy)-1,2,3-phentriazine-4-ketone (DEPBT) was
dissolved in 400 mL DMF. Then 100 mmol DIEA was added to react for 3
minutes under stirring at room temperature. The solution was added to the
resin, reacted in 37° C. water bath under N2 for 2 hours.
After the reaction, the reaction solution was drawn-off, and the resin
was washed with DMF, isopropyl alcohol (IPA), and DMF in turn.

4. Post-Processing of the Resin Peptide of Liraglutide

[0116] The resin peptide of Liraglutide obtained in step (2) was washed
with DMF, IPA and DMF in turn, and then washed three times with DCM,
washed twice with absolute ether, and dried in vacuum, to give the resin
peptide of Liraglutide.

5. Preparation of Crude Peptide of Liraglutide

[0117] The dried peptide resin of Liraglutide was reacted with fresh
lysate of trifluoroacetic acid (TFA): triisopropylsilane (TIS):
water=95:2.5:2.5 (by volume and total 10 mL of lysate per gram of the dry
resin) for 4 hours at room temperature. The reaction solution was
filtrated after the reaction, and the resin was twice washed with TFA.
The filtrate was collected, combined, and concentrated to 1/3 of the
original volume through rotary evaporation. Liraglutide was precipitated
with cold absolute ether, after centrifugation and drying under vacuum as
white crude HS-20001 is obtained.

{circle around (5)} Preparation of Liraglutide with Reversed-Phase Liquid
Chromatography

[0118] 10 g of crude Liraglutide as dissolved in a certain amount of
NH4HCO3 solution, filtrated with 0.45 μm membrane filter,
then purified with reverse-phase high performance liquid chromotagraphy
(RP-HPLC), wherein mobile phase was A 0.1% TFA/H2O, B 0.1%
TFA/acetonitrile, the column was Denali C-18 column (particle diameter
8.3 μm, 5×30 cm), column temperature was 45° C.,
detection wavelength was 220 nm, flow rate was 120 mL/min. The product
peaks were collected, concentrated with vacuum to remove most of
acetonitrile. 2.25 g of Liraglutide product was obtained by
lyophilization, the purity of which was 98%, and the yield was 12.5%.

Experimental Example 1

Testing the Agonist Activity of the Compounds on Glucagon-Like Peptide-1
Receptor (GLP1R)

[0119] GLP1R is a receptor coupled with Gs protein, of which the binding
with the agonists will result in an increase of intracellular cAMP
concentration. In the present experiment, GLP1R and the luciferase
reporter gene plasmid regulated by cAMP response elements are
co-transfected into HEK293 cells. When the compound binds to the receptor
and activates the receptors, the expression of the luciferase will
increase. The activation status of the compound to GLP1R can be learned
by testing the activity of the luciferase.

[0120] 1. HEK293 cells stably transfected with GLP1R and pCRE-Luc plasmid
were implanted in 96 well plate with the amount 40000 cells/well/100
μl, and incubated at 37° C. for 24 hours. 2. The compounds or
positive drugs having a certain concentration gradient were added (3
wells per concentration) and incubated at 37° C. for 5 hours. The
negative control was solvent DMSO. 3. 50 μl culture medium was taken
from each well, and 50 μl of the luciferase substrate were added and
vortexed for 10 minutes. 4. 80 μl reaction solution was taken and
transferred to a white 96 well plate, then detected on the Invision
microplate reader (enzyme-labelling measuring instrument).

[0121] The experimental results: compared with positive compounds
liraglutide, the activity of the compound HS-20001 is approximately equal
to that of the positive compounds, but HS-20002-20008 show much better
agonist activity.

[0122] db/db mice with type 2 diabetes were divided into six groups based
on a random blood glucose and body weight (8 per group). Physiological
saline, 3 or 10 μg/kg HS series new compounds (Liraglutide, 20001,
20002, 20003, 20004, 20005, 20006, 20007, 20008) are administered by
single subcutaneous injection. The random blood glucose of the mice is
determined at different time after administration.

[0123] The animals used in the experiment are db/db mice, which are
products of a U.S. corporation named Jackson and are conserved and
reproduced by Shanghai Institute of Materia Medica of Chinese Academy of
Science, of which the Certificate of Conformity is: SCXK(HU)2008-0017,
Body Weight: 35-50 g; Gender: Male 85, female 86, bred in SPF-grade
animal room; Temperature: 22-24° C.; Humidity: 45-80%; Light:
150-300 Lx, 12 h day alternates with night.

[0125] Preparation method: 1 bottle of the compound (2 mg/bottle) was
dissolved with double-distilled water to prepare a colorless and
transparent solution of which the concentration is 2 mg/mL. Then the
solution was diluted to 0.6 μg/mL and 2 μg/mL with physiological
saline (Sodium chloride injection, Double-Crane Pharmaceutical Co., Ltd.
Anhui, batch number: 080728 6C). "ACCU-CHEK® Advantage" blood glucose
meter form Roche was used to determine the blood glucose.

[0148] Each group has 8 mice, half male and half female. The animals of
each group were administered with the test compounds or solvent control
respectively through single subcutaneous injection. The random blood
glucose was determined at 1 h, 2 h, 4 h, 8 h and 24 h after
administration, and the decrease rate of blood glucose as calculated as
follows:

Decrease rate of blood glucose=(blood glucose of control group-blood
glucose of treatment group)/blood glucose of control group*100%.

The Experimental Results

[0149] Test 1: Effect of the Low-Dose New Compounds Administered by Singe
Dose on Random Blood Glucose of Db/Db Mice

[0150] The results can be seen in Tables 2 and 3. db/db mice were
administered with 3 μg/kg HS-20002, 20004, 20005, 20006, 20007, or
20008 through single subcutaneous injection. After one hour, random blood
glucose values of the mice were decreased significantly compared with
those of the control group (P<0.05). Decrease rates are 24.51%,
15.00%, 14.00%, 14.25%, 13.98% and 13.90% respectively. After 2 h and 4 h
from administration, random blood glucose values kept a lower level and
had significant difference from those of the control group (P<0.05).
After 8 hours from administration, random blood glucose values had no
significant difference from those of the control group. The mice were
administered with 3 μg/kg HS-20003 through subcutaneous injection.
After one hour, random blood glucose values were decreased significantly
compared with those of the control group (P<0.05); up to 17.33%, after
2 h, 4 h and 8 h from the administration, random blood glucose values
showed no significant difference from those of the control group. After
administered with 3 μg/kg HS-20001 for db/db mice through single
subcutaneous injection, random blood glucose values decreased and were
compared to those of the control group No significant difference was
observed. The values of random blood glucose of the group of mice
administered with liraglutide had no significant decrease.

[0151] Test 2: Effect of the High-Dose New Compounds Administered by
Single Dose on Random Blood Glucose of Db/Db Mice

[0152] The results can be seen in Tables 4 and 5. db/db mice were
administered with 10 μg/kg HS-20002 through single subcutaneous
injection. After one hour, the random blood glucose values of the mice
decreased significantly compared with those of the control group
(P<0.01). After 2 h, 4 h and 8 h from the administration, the random
blood glucose values kept a lower level, wherein the values at 4 h after
administration were most obvious, of which the decrease rate is up to
40.67% and is significantly different from that of the control group
(P<0.001), until 24 hours after administration, the random blood
glucose values were still significantly lower than those of the control
group. The mice were administered with 10 μg/kg HS-20003 through
single subcutaneous injection. After one hour, the random blood glucose
values were decreased significantly compared with those of the control
group (P<0.01) and is up to 23.62% decreasing, after 2 h, 4 h and 8 h
from the administration. The random blood glucose values still keep at a
lower level. After 24 hours from administration, there was no significant
difference compared with the control group. db/db mice are administered
with 10 μg/kg HS-20001 through single subcutaneous injection, after 2
h, the random blood glucose values are decreased significantly compared
with those of the control group, after 4 h and 8 h from the
administration, the random blood glucose values still keep at a lower
level. After 24 hours from administration, the random blood glucose
values showed no significant difference from those of the control group.
HS-20002, HS-20004, HS-20005, HS-20006, HS-20007 or HS-20008 were
administered to mice through single subcutaneous injection and the random
blood glucose values are decreased immediately and significantly. The
decrease rate is up to 36.20%, after 2 hours. After 4 and 8 hours from
the administration, the blood glucose values still kept at a lower level.
After 24 hours from the administration, blood glucose was not
significantly different compared with those of the control group. The
values of random blood glucose of mice of group administered with
liraglutide have no significant decrease.

[0154] The random blood glucose of db/db mice administered with series of
the new compounds of the invention through single subcutaneous injection
decreased significantly. The random blood glucose level decreased
obviously by HS-20002, HS-20003, HS-20004, HS-20005, HS-20006, HS-20007
and HS-20008 in a dose of 3 μg/kg. Where, HS-20002 and HS-20004 show a
much better effect on reducing random blood glucose, the duration of the
hypoglycemic effect after single subcutaneous injection was dose-related.
The duration of the effect of HS-20002 and HS-20004 on decreasing random
blood glucose in the dose of 3 μg/kg was more than 4 hours. The
duration of the effect of HS-20001, HS-20002, HS-20003, HS-20004,
HS-20005, HS-20006, HS-20007 and HS-20008 on decreasing random blood
glucose in the dose of 10 μg/kg was more than 8 hours.